title: A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions.
abstract: Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.
authors.affiliation: Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Du
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Nikolovska-Coleska
authors.forename: Min
authors.initials: M
authors.lastname: Qui
authors.forename: Lian
authors.initials: L
authors.lastname: Li
authors.forename: Iestyn
authors.initials: I
authors.lastname: Lewis
authors.forename: Raymond
authors.initials: R
authors.lastname: Dingledine
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Stuckey
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Krajewski
authors.forename: Peter P
authors.initials: PP
authors.lastname: Roller
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
authors.forename: Haian
authors.initials: H
authors.lastname: Fu
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay; D019411:Clinical Laboratory Techniques; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008853:Microscopy; D008904:Miniaturization; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis; D013997:Time Factors
chemical_list: D064549:Myeloid Cell Leukemia Sequence 1 Protein; C498702:PMAIP1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D054852:Small Molecule Libraries